IL-18 receptors

ABSTRACT

A novel polypeptide that functions as an IL-18 receptor is disclosed. The receptor is multimeric and includes at least one AcPL polypeptide, or fragment thereof, and at least one IL-1Rrp1 polypeptide, or fraction thereof. The receptor binds IL-18 and finds use in inhibiting biological activities mediated by IL-18.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of application U.S. Ser. No. 09/621,502,filed Jul. 21, 2000; which is a continuation of InternationalApplication number PCT/US99/01419, filed Jan. 22, 1999 and published inEnglish on Jul. 29, 1999; which claims the benefit under 35 U.S.C.§119(e) of U.S. Provisional Applications Ser. No. 60/072,301 filed Jan.23, 1998; Ser. No. 60/078,835 filed Mar. 20, 1998; and Ser. No.60/094,469 filed Jul. 28, 1998, all of which are incorporated byreference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to proteins that are members of the IL-1receptor family. More particularly, the present invention relates toIL-1Rp1 and AcPL receptor complexes that mediate high affinity IL-18binding and activity as well as inhibit IL-18 mediated activity.

2. Description of Related Art

The type I interleukin-1 receptor (IL-1RI) mediates the biologicaleffects of interleukin-1, a pro-inflammatory cytokine (Sims et al.,Science 241:585-589, 1988; Curtis et al., Proc. Natl. Acad. Sci. USA86:3045-3049, 1989). A second interleukin-1 receptor (designated type IIIL-1R or IL-1RII) binds IL-1, but does not appear to mediate signaltransduction (McMahan et al., EMBO J. 10:2821, 1991; Sims et al., Proc.Natl. Acad. Sci. USA 90:6155-6159, 1993). IL-1RI and IL-1RII each bindIL-1□ and IL-1β. IL-1 has been implicated in chronic inflammatorydiseases, such as rheumatoid arthritis and inflammatory bowel disease.There is increasing evidence that IL-1 plays a role in osteoporosis. Allof these activities are initiated by the signaling function of thecytoplasmic portion of the Type I IL-1R. IL-1ra inhibits the activitiesof IL-1 by binding to the type I IL-1 receptor, thereby blocking accessto IL-1α and IL-1β while eliciting no biological response of its own.

IL-1RI and IL-1RII belong to a family of proteins that exhibitsignificant sequence homology. One such protein is IL-1R accessoryprotein (IL-1R AcP), described in Greenfeder et al. (J. Biol. Chem. 270:13757-13765, 1995). This protein, by itself, is not capable of bindingIL-1, but does form a complex with IL-1RI and IL-1α and IL-1β. Whenco-expressed with IL-1RI, recombinant IL-1R AcP increases the bindingaffinity of IL-1RI for IL-1β (Greenfeder et al., supra).

Another protein exhibiting sequence homology to the IL-1RI and IL-1RIIfamily is the IL-1 receptor related protein 1 (IL-1Rrp1) (See Parnet etal. J. Biol Chem 271:3967, 1996, and Torigoe et al., J. Biol Chem272:25737, 1997). Still another such protein is AcPL.

IL-18 is a homologue of IL-1α and IL-1β and is known to activate many ofthe same responses activated by IL-1. For example, cells stimulated withIL-18 activate NFκB and produce known inflammatory mediators. IL-18 actsas a stimulator of Th1 cell growth and differentiation and is a potentinducer of γ-interferon production from Th1 cells. The Th1 class ofhelper T cells are known to mediate inflammatory reactions. IL-18enhances NK cell killing activity and has been implicated in septicshock, liver destruction, inflammatory bowel disease and diabetes.

Recently it was shown that IL-1Rrp1 binds IL-18 and mediates IL-18signaling in transfected cells. However, the IL-1Rrp1 binding affinityfor IL-18 is very low and it is likely that one or more additionalreceptors or receptor subunits are involved with IL-18 binding andsignaling.

Thus, the identification of additional receptors of for IL-18 isdesirable. Such receptor proteins can be studied to determine whether ornot they bind IL-18 and, if so, whether the receptors play a role inmediating signal transduction. Furthermore, soluble forms of suchreceptors may be used to inhibit IL-18 activity and ameliorate anyinflammatory and/or autoimmune diseases attributable to IL-18 signaling.The possible existence of additional affinity-converting subunits forIL-18 can be explored, as well.

SUMMARY OF THE INVENTION

The present invention provides receptor polypeptides designated hereinas IL-18 receptor complexes. More particularly, the present inventionprovides multimeric receptor polypeptides that include an AcPLpolypeptide, or fragments thereof, and an IL-1Rrp1 polypeptide, orfragments thereof. The AcPL polypeptide may be covalently linked ornoncovalently to the IL-1Rrp1 polypeptide by any suitable means. Suchmeans include via a cross-linking reagent, a polypeptide linker, andassociations such as via disulfide bonds or by use of leucine zippers.In one embodiment of the invention, the receptor is a fusion proteinproduced by recombinant DNA technology. This multimeric receptor of thepresent invention binds IL-18 with an affinity greater than that ofIL-1Rrp1 alone. Disorders mediated by IL-18 may be treated byadministering a therapeutically effective amount of this inventivereceptor to a patient afflicted with such a disorder.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based upon the discovery that the coexpressionof AcPL and IL-1Rrp1 results in a dramatic enhancement of NFκB activityin cells stimulated with IL-18. Because IL-1Rrp1 alone binds IL-18 onlyweakly and AcPL does not bind IL-18, the enhancement of NFκB activity bycoexpressed AcPL and IL-1Rrp1 indicates that these polypeptides aresubunits of an IL-18 receptor complex. In accordance with the presentinvention novel polypeptides, designated IL-18 receptor complexes, areprovided. Advantageously, such dimeric IL-18 receptor complexescomprising IL-1Rrp1 and AcPL, or fragments thereof, are useful forinhibiting IL-18 activity, including the proinflammatory effects ofIL-18, and can include IL-1Rrp1 and AcPL as proteins coexpressed in thesame cell, or as IL-1Rrp1 linked to an AcPL as receptor subunits.Preferably the subunits are linked via covalent linkages. The subunitsmay be covalently linked by any suitable means, such as via across-linking reagent or a polypeptide linker.

In one embodiment of the present invention, the receptor is a fusionprotein produced by recombinant DNA technology. Such fusion proteins canbe prepared by transfecting cells with DNA encoding IL-1Rrp1:Fc fusionprotein and DNA encoding AcPL:Fc fusion protein and coexpressing thedimers in the same cells.

Alternatively, AcPL/IL-1Rrp1 dimers can be prepared by fusing one of thereceptor subunits to the constant region of an immunoglobulin heavychain and fusing the other receptor subunit to the constant region of animmunoglobulin light chain. For example, an AcPL protein can be fused tothe CH₁-hinge-CH₂—CH₃ region of human IgG1 and an IL-1Rrp1 protein canbe fused to the C kappa region of the Ig kappa light chain, or viceversa. Cells transfected with DNA encoding the immunoglobulin lightchain fusion protein and the immunoglobulin heavy chain fusion proteinexpress heavy chain/light chain heterodimers containing the AcPL fusionprotein and the IL-1Rrp1 fusion protein. Via disulfide linkages betweenthe heavy chains, the heterodimers further combine to provide multimers,largely tetramers. Advantageously, in the event homodimers of two heavyor two light chain fusions are expressed, such homodimers can beseparated easily from the heterodimers.

In addition to IL-18 receptor protein complexes, the present inventionincludes isolated DNA encoding heteromer polypeptides, expressionvectors containing DNA encoding the heteromer polypeptides, and hostcells transformed with such expression vectors. Methods for productionof recombinant IL-18 receptor, including soluble forms of the protein,are also disclosed. Antibodies immunoreactive with the novel polypeptideare provided herein as well.

In one embodiment of the present invention, the polypeptide subunits ofthe heteromer IL-18 receptors include at least one AcPL subunit asdescribed in SEQ ID NO:2 or SEQ ID NO: 6, and at least one IL-1Rrp1subunit as described in SEQ ID NO:4 or SEQ ID NO:8. DNA encoding thesepolypeptide subunits are presented in SEQ ID NO:1, SEQ ID NO:5, SEQ IDNO:3 and SEQ ID NO:7, respectively. The AcPL subunit protein encoded bySEQ ID NO:1 includes an extracellular domain of 356 amino acids(residues 1-356 from N- to C-terminus of SEQ ID NO:2) that includes asignal peptide of 14 amino acids (residues 1-14 of SEQ ID NO:2); atransmembrane region of 25 amino acids (residues 357-381) and acytoplasmic domain of 218 amino acids (residues 382-599). The AcPLsubunit protein encoded by SEQ ID NO:5 includes an extracellular domainof 356 amino acids (residues 1-356 of SEQ ID NO:6) that includes asignal peptide of 14 amino acids (residues 1-14 of SEQ ID NO:6); atransmembrane region of 24 amino acids (residues 357-380) and acytoplasmic domain of amino acid residues 381-614. The IL-1Rrp1 subunitprotein encoded by SEQ ID NO:3 includes an extracellular domain of 329amino acids (residues 1-329 of SEQ ID NO:4) that includes a signalpeptide of 19 amino acids (residues 1-19 of SEQ ID NO:4); atransmembrane region of 21 amino acids (residues 330 to 350 of SEQ IDNO:4); and, a cytoplasmic domain from amino acid residues 351 to 541.The IL-1Rrp1 subunit protein encoded by SEQ ID NO:7 includes anextracellular domain of 322 amino acids (residues 1-322 of SEQ ID NO:8)that includes a signal peptide of 18 amino acids (residues 1-18 of SEQID NO:8); a transmembrane region of 25 amino acids (residues 323 to 347of SEQ ID NO:8); and, a cytoplasmic domain from amino acid residues 348to 537. Additionally, IL-1Rrp1 is described in U.S. Pat. No. 5,776,731and AcPL is described in copending applications Ser. No. 60/078,835 andSer. No. 60/072,301, incorporated herein by reference.

Preferably the polypeptide subunits of the dimeric IL-18 receptors aresoluble fragments of IL-1Rrp1 and AcPL polypeptides which together formheteromer complexes having the desired activity. Such polypeptidesinclude those lacking all or part of the transmembrane region and thecytoplasmic domain of the protein. Thus, for example, a heteromerreceptor complex of the present invention can include at least onesubunit that is the extracellular domain of SEQ ID NO:2 or SEQ ID NO:6and at least one subunit that is the extracellular domain of SEQ ID NO:4or SEQ ID NO:8. These soluble extracellular domains of AcPL and IL-1Rrp1can include or exclude their signal peptide. Thus, in anotherembodiment, a heteromeric IL-18 receptor includes amino acid residues1-356 or residues 15-356 of SEQ ID NO:2 or SEQ ID NO:6, and amino acidresidues 1-329 or residues 20-329 of SEQ ID NO:4, or amino acid residues1-325 or residues 19-322 of SEQ ID NO:8. The desirability of includingthe signal sequence depends on such factors as the position of the AcPLor IL-1Rrp1 in the fusion protein and the intended host cells when thereceptor is to be produced via recombinant DNA technology. In preferredembodiments, a DNA construct encoding one of the soluble AcPL or solubleIL-1Rrp1 fragments is fused to a DNA construct encoding the constantregion of an immunoglobulin heavy chain and a DNA construct encoding theother of the soluble AcPL or soluble IL-1Rrp1 fragment is fused to DNAencoding the constant region of an immunoglobulin light chain.

Alternatively, the IL-18 receptor may comprise IL-1Rrp1 or solubleIL-1Rrp1 fragments non-covalently complexed with AcPL or soluble AcPLfragments. Non-covalent bonding of IL-1Rrp1 to AcPL may be achieved byany suitable means that does not interfere with the receptor's abilityto bind IL-18. In one approach, a first compound is attached to IL-1Rrp1and a second compound that will non-covalently bond to the firstcompound is attached to AcPL. Examples of such compounds are biotin andavidin. The receptor is thus formed through the non-covalentinteractions of biotin with avidin. In one embodiment of the invention,IL-1Rrp1 and AcPL are recombinant polypeptides, each purified fromrecombinant cells and then non-covalently bonded together to form thereceptor. A host cell may be transformed with two different expressionvectors such that both IL-1Rrp1 and AcPL are produced by the recombinanthost cell. IL-1Rrp1 and AcPL produced by such transformed host cells mayassociate to form a complex through non-covalent interactions. When suchtransformed cells express the membrane-bound forms of the proteins, suchcells are useful in various assays, including competition assays.

The binding assay described in example 1 compares the binding of IL-18by supernatant from cells transfected with IL-1Rrp1 alone, AcPL aloneand a combination of IL-1Rrp1 and AcPL. Supernatants from cellscoexpressing IL-1Rrp1 and AcPL exhibited high levels of IL-18 binding;supernatants from cells expressing IL-1Rrp1 alone exhibited low levelsof IL-18 binding; and, supernatant from cells transfected with AcPLalone do not bind IL-18. The NFκB induction assay described in example 2demonstrates that cells transfected with IL-1Rrp1 alone and cellstransfected with AcPL alone are not responsive to IL-18 stimulation.However, cells co-transfected with both IL-1Rrp1 and AcPL and stimulatedwith IL-18 greatly enhanced NFκB induction.

As used herein, the terms IL-1Rrp1 and AcPL include variants andtruncated forms of the native proteins that possess the desiredbiological activity. Variants produced by adding, substituting, ordeleting amino acid(s) in the native sequence are discussed in moredetail below.

As described above, soluble IL-1Rrp1 and soluble AcPL polypeptides arepreferred for certain applications. “Soluble IL-1Rrp1” as used in thecontext of the present invention refers to polypeptides that aresubstantially similar in amino acid sequence to all or part of theextracellular region of a native IL-1Rrp1 polypeptide and that, due tothe lack of a transmembrane region that would cause retention of thepolypeptide on a cell membrane, are secreted upon expression. Suitablesoluble IL-1Rrp1 polypeptides retain the desired biological activity.Soluble IL-1Rrp1 may also include part of the transmembrane region orpart of the cytoplasmic domain or other sequences, provided that thesoluble IL-1Rrp1 protein is capable of being secreted.

Likewise, the term “soluble AcPL” as used herein refers to proteins thatare substantially similar in amino acid sequence to all or part of theextracellular region of a native AcPL polypeptide and are secreted uponexpression but retain the desired biological activity. Soluble AcPL mayinclude part of the transmembrane region, cytoplasmic domain, or othersequences, as long as the polypeptide is secreted.

In one embodiment, soluble IL-1Rrp1 and AcPL polypeptides include theentire extracellular domain. To effect secretion, the solublepolypeptides comprise the native signal peptide or a heterologous signalpeptide. Thus, examples of soluble IL-1Rrp1 polypeptides comprise aminoacids 1-329 of SEQ ID NO:4 (human IL-1Rrp1) and amino acids 1-322 of SEQID NO:8 (murine IL-1Rrp1). Examples of soluble AcPL polypeptidescomprise amino acids 1-356 of SEQ ID NO:2 (human AcPL) and amino acids1-356 of SEQ ID NO:6 (murine AcPL).

A soluble fusion protein comprising the extracellular domain of IL-1Rrp1of SEQ ID NO:4 fused to an antibody Fc region polypeptide and theextracellular domain of AcPL fused to an Fc region polypeptide, isdescribed in example 1.

Soluble AcPL and soluble IL-1Rrp1 may be identified (and distinguishedfrom their non-soluble membrane-bound counterparts) by separating intactcells which express the desired protein from the culture medium, e.g.,by centrifugation, and assaying the medium (supernatant) for thepresence of the desired protein. The culture medium may be assayed usingprocedures which are similar or identical to those described in theexamples below. The presence of AcPL or IL-1Rrp1 in the medium indicatesthat the protein was secreted from the cells and thus is a soluble formof the desired protein. Soluble AcPL and soluble IL-1Rrp1 may benaturally-occurring forms of these proteins. Alternatively, solublefragments of AcPL and IL-1Rrp1 proteins may be produced by recombinantDNA technology or otherwise isolated, as described below.

The use of soluble forms of IL-1Rrp1 and AcPL is advantageous forcertain applications. Purification of the proteins from recombinant hostcells is facilitated, since the soluble proteins are secreted from thecells. Further, a receptor of the present invention comprising solubleIL-1Rrp1 and AcPL proteins is generally more suitable for intravenousadministration.

With respect to the foregoing discussion of signal peptides and thevarious domains of the IL-1Rrp1 and AcPL proteins, the skilled artisanwill recognize that the above-described boundaries of such regions ofthe proteins are approximate. For example, although computer programsthat predict the site of cleavage of a signal peptide are available,cleavage can occur at sites other than those predicted. Further, it isrecognized that a protein preparation can comprise a mixture of proteinmolecules having different N-terminal amino acids, due to cleavage ofthe signal peptide at more than one site. Thus, soluble IL-1Rrp1 andAcPL polypeptides comprising the extracellular domain include thosehaving a C-terminal amino acid that may vary from that identified aboveas the C-terminus of the extracellular domain. Further,post-translational processing that can vary according to the particularexpression system employed may yield proteins having differingN-termini. Such variants that retain the desired biological activitiesare encompassed by the terms “IL-1Rrp1 polypeptides” and “AcPLpolypeptides” as used herein.

Truncated IL-1Rrp1 and AcPL, including soluble polypeptides, may beprepared by any of a number of conventional techniques. In the case ofrecombinant proteins, a DNA fragment encoding a desired fragment may besubcloned into an expression vector. Alternatively, a desired DNAsequence may be chemically synthesized using known techniques. DNAfragments also may be produced by restriction endonuclease digestion ofa full length cloned DNA sequence, and isolated by electrophoresis onagarose gels. Linkers containing restriction endonuclease cleavagesite(s) may be employed to insert the desired DNA fragment into anexpression vector, or the fragment may be digested at cleavage sitesnaturally present therein. Oligonucleotides that reconstruct the N- orC-terminus of a DNA fragment to a desired point may be synthesized. Theoligonucleotide may contain a restriction endonuclease cleavage siteupstream of the desired coding sequence and position an initiation codon(ATG) at the N-terminus of the coding sequence.

The well known polymerase chain reaction procedure also may be employedto isolate a DNA sequence encoding a desired protein fragment.Oligonucleotide primers comprising the desired termini of the fragmentare employed in such a polymerase chain reaction. Any suitable PCRprocedure may be employed. One such procedure is described in Saiki etal., Science 239:487 (1988). Another is described in Recombinant DNAMethodology, Wu et al., eds., Academic Press Inc., San Diego (1989), pp.189-196. In general, PCR reactions involve combining the 5′ and 3′oligonucleotide primers with template DNA (in this case, IL-1Rrp1 orAcPL DNA) and each of the four deoxynucleoside triphosphates, in asuitable buffered solution. The solution is heated, (e.g., from 95° C.to 100° C.) to denature the double-stranded DNA template and is thencooled before addition of a DNA polymerase enzyme. Multiple cycles ofthe reactions are carried out in order to amplify the desired DNAfragment.

The AcPL polypeptide is attached to the IL-1Rrp1 polypeptide through acovalent or non-covalent linkage. Covalent attachment is preferred forcertain applications, e.g. in vivo use, in view of the enhancedstability generally conferred by covalent, as opposed to non-covalent,bonds. In constructing the receptor of the present invention, covalentlinkage may be accomplished via cross-linking reagents, peptide linkers,or any other suitable technique.

Numerous reagents useful for cross-linking one protein molecule toanother are known. Heterobifunctional and homobifunctional linkers areavailable for this purpose from Pierce Chemical Company, Rockford, Ill.,for example. Such linkers contain two functional groups (e.g., estersand/or maleimides) that will react with certain functional groups onamino acid side chains, thus linking one polypeptide to another.

One type of peptide linker that may be employed in the present inventionseparates AcPL and the IL-1Rrp1 domains by a distance sufficient toensure that each domain properly folds into the secondary and tertiarystructures necessary for the desired biological activity. The linkeralso should allow the extracellular domains of AcPL and IL-1Rrp1 toassume the proper spatial orientation to form the binding site forIL-18.

Suitable peptide linkers are known in the art, and may be employedaccording to conventional techniques. Among the suitable peptide linkersare those described in U.S. Pat. Nos. 4,751,180 and 4,935,233, which arehereby incorporated by reference. A peptide linker may be attached to byany of the conventional procedures used to attach one polypeptide toanother. The cross-linking reagents available from Pierce ChemicalCompany as described above are among those that may be employed. Aminoacids having side chains reactive with such reagents may be included inthe peptide linker, e.g., at the termini thereof. Preferably, a fusionprotein comprising AcPL joined to IL-1Rrp1 via a peptide linker isprepared by recombinant DNA technology.

In one embodiment of the invention, AcPL and IL-1Rrp1 are linked viapolypeptides derived from immunoglobulins. Preparation of fusionproteins comprising heterologous polypeptides fused to various portionsof antibody-derived polypeptides (including the Fc domain) has beendescribed, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991) and Byrnet al. (Nature 344:677, 1990). As one example, a polypeptide derivedfrom the Fc region of an antibody may be attached to the C-terminus ofIL-1Rrp1. A separate Fc polypeptide is attached to the C-terminus ofAcPL. Disulfide bonds form between the two Fc polypeptides (e.g., in theso-called hinge region, where interchain disulfide bonds are normallypresent in antibody molecules), producing a heterodimer comprising theAcPL/Fc fusion protein linked to the IL-1Rrp1/Fc fusion protein.Advantageously, host cells are co-transfected with two differentexpression vectors, one encoding soluble IL-1Rrp1/Fc and the otherencoding soluble AcPL/Fc. The heterodimer is believed to formintracellularly or during secretion.

The term “Fc polypeptide” as used herein includes native and muteinforms, as well as truncated Fc polypeptides containing the hinge regionthat promotes dimerization. cDNA encoding a single chain polypeptidederived from the Fc region of a human IgG1 antibody can be cloned intothe pBluescript SK® cloning vector (Stratagene Cloning Systems, LaJolla,Calif.) to produce a recombinant vector designated hIgG1Fc. A uniqueBglII site is positioned near the 5′ end of the inserted Fc encodingsequence. An SpeI site is immediately downstream of the stop codon. TheFc polypeptide encoded by the cDNA extends from the N-terminal hingeregion to the native C-terminus, i.e., is an essentially full-lengthantibody Fc region. One suitable mutein of this Fc polypeptide isdescribed in U.S. patent application Ser. No. 08/097,827, herebyincorporated by reference. The mutein exhibits reduced affinity for Fcreceptors.

Homodimers comprising two IL-1Rrp1/Fc polypeptides or two AcPL/Fcpolypeptides linked via disulfide bonds are also produced by certain ofthe transfected host cells disclosed herein. The homodimers may beseparated from each other and from the heterodimer by virtue ofdifferences in size (e.g., by gel electrophoresis). The heterodimer alsomay be purified by sequential immunoaffinity chromatography (describedbelow).

Il-18 receptor complexes of the present invention include fusionproteins of the constant region of an antibody light chain (or fragmentthereof) and the constant region of an antibody heavy chain (or afragment thereof). The constant region of the heavy chain can includeall four of its constant region domains or portion of the domains,including the CH₁ which associates with the light chain, the H hingeregion, and the CH₂ and CH₃ domains which are responsible for thedimerization of the heavy chain molecules. Within the scope of theforegoing fusion proteins are tetramers that are formed by two dimerswhich link heavy chain/light chain dimers via disulfide linkages betweentheir respective heavy chain regions.

With respect to immunoglobulin light chain polypeptides, polypeptides ofthe κ family and the λ family are suitable in the practice of thisinvention. Thus, any type of immunoglobulin dimers or tetramer includingIgM, IgD, IgG, IgA and IgE can be the basis of the heteromer moleculesof the present invention.

In accordance with the present invention, functional heteromericpolypeptides can be prepared by the association between multiple heavyand multiple light chain molecules which normally associate with oneanother. For example, the constant region of human IgG1 will associatewith the constant region of human light chain kappa (designatedC_(kappa)). The amino acid sequence of hIgG1 constant region has beenreported (Ellison, J W, Berson, B J and Hood, L E 1982). The nucleotidesequence of a human immunoglobulin C gamma 1 gene is reported. (Nuc.Acids Res. 10: 4071 and Walls, M A, Hsiao, K C and Harris, L J 1993).Vectors for the expression of PCR-amplified immunoglobulin variabledomains with human constant regions are disclosed. (Nuc. Acids Res.21:2921) The sequence of human light chain C_(kappa) has also beenreported (Shuford, W, Raff, H V, Finley, J W, Esselstyn, J and Harris, LJ. 1991) Effect of light chain V-region duplication on IgGoligomerization and in vivo efficacy. Science 252:724 and Steinberger,P, Kraft, D and Valenta, R (1996). Construction of a combinatorial IgElibrary from an allergic patient. Isolation and characterization ofhuman IgE Fabs with specificity for the major timothy grass pollenallergen, Ph1 p 5. J. Biol. Chem., 271:10972).

IL-18 receptor embodiments that include heavy and light chain antibodyregions are fusion proteins represented by the formulae:R₁-L₁:R₂-₋L₂ or R₂-L₂:R₁-L₁ or R₁-L₂:R₂-L₁ or R₂-L₁:R₁-L₂R₁-L₁:R₂₋-L₂/R₂-L₂:R₁-L₁ or R₁-L₂:R₂-L₁/R₂-L₁:R₁-L₂in which L₁ is an immunoglobulin heavy chain fragment, the N terminus ofwhich extends at least through the C_(H)1 region; L₂ is animmunoglobulin light chain fragment; R₁ is AcPL or an AcPL fragment; R₂is IL-1Rrp1 or an IL-1Rrp1 fragment;: designates linkages between aheavy chain and light chain antibody region, and/designates linkagesbetween a heavy chain and a heavy chain antibody region. In the case ofa dimer, the resulting fusion polypeptide includes two receptor subunitsjoined by a heavy chain/light chain. In the case of the tetramer, thefusion protein includes four receptor subunits and resembles an antibodyin structure, displaying the IL-18 binding site bivalently.

To obtain the foregoing fusion polypeptides, cDNA encoding an antibodyheavy chain polypeptide derived from human IgG1 antibody (CH₁—H—CH₂—CH₃)can be cloned into the pDC409 expression vector to produce a recombinantvector designated hIgG1. A unique BglII site is positioned near the 5′end of the inserted heavy chain encoding sequence. A NotI site isimmediately downstream of the stop codon. The heavy chain polypeptide,encoded by the cDNA extends from the N-terminus of the CH₁ region to thenative C-terminus. To obtain an antibody light chain cDNA encoding asingle chain polypeptide derived from the human kappa chain constantregions can be cloned in the pDC409 expression vector to produce arecombinant vector designated hIgκ. This sequence is flanked at the 5′end by a unique BglII site and at the 3′ end by a unique NotI site.Embodiments of the present invention that incorporate such antibodypolypeptides include a first fusion polypeptide comprising AcPL (or afragment thereof) upstream of the constant region of an antibody lightchain (or a fragment thereof) and a second fusion polypeptide comprisingIL-1Rrp1 upstream of the constant region of an antibody heavy chain (ora heavy chain fragment), the N-terminus of which extends at leastthrough the C_(H)1 region. Disulfide bond(s) form between the AcPL lightchain fusion polypeptide and the IL-1Rrp1-heavy chain fusionpolypeptide, thus producing a receptor of the present invention. As afurther alternative, an IL-1Rrp1-antibody light chain fusion polypeptideis prepared and combined with (disulfide bonded to) a fusion polypeptidecomprising AcPL-antibody heavy chain fusion polypeptide. When two of theforegoing disulfide bonded molecules are combined, additional disulfidebonds form between the two antibody regions. The resulting receptor ofthe present invention comprising four fusion polypeptides resembles anantibody in structure and displays the IL-18 binding site bivalently.

The AcPL and IL-1Rrp1 polypeptides may be separately purified fromcellular sources, and then linked together. Alternatively, the receptorof the present invention may be produced using recombinant DNAtechnology. The AcPL and IL-1Rrp1 polypeptides may be producedseparately and purified from transformed host cells for subsequentcovalent linkage. In one embodiment of the present invention, a hostcell is transformed/transfected with foreign DNA that encodes AcPL andIL-1Rrp1 as separate polypeptides. The two polypeptides may be encodedby the same expression vector with start and stop codons for each of thetwo genes, or the recombinant cells may be co-transfected with twoseparate expression vectors. In another embodiment, the receptor isproduced as a fusion protein in recombinant cells.

In one embodiment of the present invention, the receptor protein is arecombinant fusion protein of the formula:R₁-L-R₂ or R₂-L-R₁wherein R₁ represents AcPL or an AcPL fragment; R₂ represents IL-1Rrp1or an IL-1Rrp1 fragment; and L represents a peptide linker.

The fusion proteins of the present invention include constructs in whichthe C-terminal portion of AcPL is fused to the linker which is fused tothe N-terminal portion of IL-1Rrp1, and also constructs in which theC-terminal portion of IL-1Rrp1 is fused to the linker which is fused tothe N-terminal portion of AcPL. AcPL is covalently linked to IL-1Rrp1 insuch a manner as to produce a single protein which retains the desiredbiological activities of AcPL and IL-1Rrp1. The components of the fusionprotein are listed in their order of occurrence (i.e., the N-terminalpolypeptide is listed first, followed by the linker and then theC-terminal polypeptide).

A DNA sequence encoding a fusion protein is constructed usingrecombinant DNA techniques to insert separate DNA fragments encodingAcPL and IL-1Rrp1 into an appropriate expression vector. The 3′ end of aDNA fragment encoding AcPL is ligated (via the linker) to the 5′ end ofthe DNA fragment encoding IL-1Rrp1 with the reading frames of thesequences in phase to permit translation of the mRNA into a singlebiologically active fusion protein. Alternatively, the 3′ end of a DNAfragment encoding IL-1Rrp1 may be ligated (via the linker) to the 5′ endof the DNA fragment encoding AcPL, with the reading frames of thesequences in phase to permit translation of the mRNA into a singlebiologically active fusion protein. A DNA sequence encoding anN-terminal signal sequence may be retained on the DNA sequence encodingthe N-terminal polypeptide, while stop codons, which would preventread-through to the second (C-terminal) DNA sequence, are eliminated.Conversely, a stop codon required to end translation is retained on thesecond DNA sequence. DNA encoding a signal sequence is preferablyremoved from the DNA sequence encoding the C-terminal polypeptide.

A DNA sequence encoding a desired polypeptide linker may be insertedbetween, and in the same reading frame as, the DNA sequences encodingAcPL and IL-1Rrp1 using any suitable conventional technique. Forexample, a chemically synthesized oligonucleotide encoding the linkerand containing appropriate restriction endonuclease cleavage sites maybe ligated between the sequences encoding AcPL and IL-1Rrp1.

Alternatively, a chemically synthesized DNA sequence may contain asequence complementary to the 3′ terminus (without the stop codon) ofeither AcPL and IL-1Rrp1, followed by a linker-encoding sequence whichis followed by a sequence complementary to the 5′ terminus of the otherof AcPL and IL-1Rrp1. Oligonucleotide directed mutagenesis is thenemployed to insert the linker-encoding sequence into a vector containinga direct fusion of AcPL and IL-1Rrp1.

The present invention provides isolated DNA sequences encoding theabove-described fusion proteins comprising AcPL, IL-1Rrp1, and a peptidelinker. DNA encoding AcPL polypeptides disclosed herein is alsoprovided, as is DNA encoding AcPL polypeptides fused toimmunoglobulin-derived polypeptides. AcPL-encoding DNA encompassed bythe present invention includes, for example, cDNA, chemicallysynthesized DNA, DNA isolated by PCR, genomic DNA, and combinationsthereof.

Also provided herein are recombinant expression vectors containing theisolated DNA sequences. “Expression vector” refers to a replicable DNAconstruct used to express DNA which encodes the desired protein andwhich includes a transcriptional unit comprising an assembly of (1)genetic element(s) having a regulatory role in gene expression, forexample, promoters, operators, or enhancers, operatively linked to (2) aDNA sequence encoding a desired protein which is transcribed into mRNAand translated into protein, and (3) appropriate transcription andtranslation initiation and termination sequences. The choice of promoterand other regulatory elements generally varies according to the intendedhost cell.

In the expression vectors, regulatory elements controlling transcriptionor translation are generally derived from mammalian, microbial, viral orinsect genes. The ability to replicate in a host, usually conferred byan origin of replication, and a selection gene to facilitate recognitionof transformants may additionally be incorporated. Vectors derived fromretroviruses also may be employed.

DNA regions are operably linked when they are functionally related toeach other. For example, DNA encoding a signal peptide (secretoryleader) is operably linked to DNA for a polypeptide if the polypeptideis expressed as a precursor that is secreted through the host cellmembrane; a promoter is operably linked to a coding sequence if itcontrols the transcription of the sequence; and a ribosome binding siteis operably linked to a coding sequence if it is positioned so as topermit translation. Generally, “operably linked” means contiguous and,in the case of secretory leaders, contiguous and in reading frame.

Transformed host cells are cells which have been transformed ortransfected with foreign DNA using recombinant DNA techniques. In thecontext of the present invention, the foreign DNA includes a sequenceencoding the inventive proteins. Host cells may be transformed forpurposes of cloning or amplifying the foreign DNA, or may be transformedwith an expression vector for production of the protein. Suitable hostcells include prokaryotes, yeast or higher eukaryotic cells. Appropriatecloning and expression vectors for use with bacterial, fungal, yeast,and mammalian cellular hosts are described by Pouwels et al. (CloningVectors: A Laboratory Manual, Elsevier, New York, 1985), the relevantdisclosure of which is hereby incorporated by reference.

Prokaryotes include gram negative or gram positive organisms, forexample E. coli or bacilli. Prokaryotic expression vectors generallycomprise one or more phenotypic selectable markers, for example a geneencoding proteins conferring antibiotic resistance or supplying anautotrophic requirement, and an origin of replication recognized by thehost to ensure amplification within the host. Examples of suitableprokaryotic hosts for transformation include E. coli, Bacillus subtilis,Salmonella typhimurium, and various species within the generaPseudomonas, Streptomyces, and Staphylococcus, although others may alsobe employed as a matter of choice.

Useful expression vectors for bacterial use can comprise a selectablemarker and bacterial origin of replication derived from commerciallyavailable plasmids comprising genetic elements of the well-known cloningvector pBR322 (ATCC 37017). Such commercial vectors include, forexample, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1(Promega Biotec, Madison, Wis., USA). These pBR322 “backbone” sectionsare combined with an appropriate promoter and the structural sequence tobe expressed. E. coli is typically transformed using derivatives ofpBR322, a plasmid derived from an E. coli species (Bolivar et al., Gene2:95, 1977). pBR322 contains genes for ampicillin and tetracyclineresistance and this provides simple means for identifying transformedcells.

Promoters commonly used in recombinant microbial expression vectorsinclude the β-lactamase (penicillinase) and lactose promoter system(Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544,1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. AcidsRes. 8:4057, 1980; and EPA 36,776) and tac promoter (Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412,1982). A particularly useful bacterial expression system employs thephage λ P_(L) promoter and cI857ts thermoinducible repressor. Plasmidvectors available from the American Type Culture Collection whichincorporate derivatives of the λ P_(L) promoter include plasmid pHUB2,resident in E. coli strain JMB9 (ATCC 37092) and pPLc28, resident in E.coli RR1 (ATCC 53082).

The recombinant receptor protein may also be expressed in yeast hosts,preferably from Saccharomyces species, such as S. cerevisiae. Yeast ofother genera such as Pichia or Kluyveromyces may also be employed. Yeastvectors will generally contain an origin of replication from the 2 μmyeast plasmid or an autonomously replicating sequence (ARS), a promoter,DNA encoding the receptor fusion protein, sequences for polyadenylationand transcription termination and a selection gene. Preferably, yeastvectors will include an origin of replication and selectable markerspermitting transformation of both yeast and E. coli, e.g., theampicillin resistance gene of E. coli and the S. cerevisiae trp1 gene,which provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, and a promoter derived from a highlyexpressed yeast gene to induce transcription of a structural sequencedownstream. The presence of the trp1 lesion in the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

Suitable promoter sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978),such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described in R.Hitzeman et al., EPA 73,657.

Preferred yeast vectors can be assembled using DNA sequences from pBR322for selection and replication in E. coli (Amp^(r) gene and origin ofreplication) and yeast DNA sequences including a glucose-repressibleADH2 promoter and α-factor secretion leader. The ADH2 promoter has beendescribed by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier etal., (Nature 300:724, 1982). The yeast α-factor leader, which directssecretion of heterologous proteins, can be inserted between the promoterand the structural gene to be expressed. See, e.g., Kurjan et al., Cell30:922, 1982; and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330,1984. The leader sequence may be modified to contain, near its 3′ end,one or more useful restriction sites to facilitate fusion of the leadersequence to foreign genes.

Suitable yeast transformation protocols are known to those of skill inthe art. An exemplary technique is described by Hinnen et al., Proc.Natl. Acad. Sci. USA 75:1929, (1978), selecting for Trp⁺ transformantsin a selective medium consisting of 0.67% yeast nitrogen base, 0.5%casamino acids, 2% glucose, 10 μg/ml adenine and 20 μg/ml uracil.

Host strains transformed by vectors comprising the ADH2 promoter may begrown for expression in a rich medium consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 μg/ml adenine and 80 μg/mluracil. Derepression of the ADH2 promoter occurs upon exhaustion ofmedium glucose. Crude yeast supernatants are harvested by filtration andheld at 4° C. prior to further purification.

Various mammalian or insect cell culture systems can be employed toexpress recombinant protein. Baculovirus systems for production ofheterologous proteins in insect cells are reviewed by Luckow andSummers, Bio/Technology 6:47 (1988). Examples of suitable mammalian hostcell lines include L cells, C127, 3T3, Chinese hamster ovary (CHO),HeLa, and BHK cell lines. Additional suitable mammalian host cellsinclude CV-1 cells (ATCC CCL70) and COS-7 cells (ATCC CRL 1651;described by Gluzman, Cell 23:175, 1981), both derived from monkeykidney. Another monkey kidney cell line, CV-1/EBNA (ATCC CRL 10478), wasderived by transfection of the CV-1 cell line with a gene encodingEpstein-Barr virus nuclear antigen-1 (EBNA-1) and with a vectorcontaining CMV regulatory sequences (McMahan et al., EMBO J. 10:2821,1991). The EBNA-1 gene allows for episomal replication of expressionvectors, such as HAV-EO or pDC406, that contain the EBV origin ofreplication.

Mammalian expression vectors may comprise non-transcribed elements suchas an origin of replication, a suitable promoter and enhancer linked tothe gene to be expressed, and other 5′ or 3′ flanking nontranscribedsequences, and 5′ or 3′ nontranslated sequences, such as necessaryribosome binding sites, a poly-adenylation site, splice donor andacceptor sites, and transcriptional termination sequences. Thetranscriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. For example, commonly used promoters and enhancers arederived from Polyoma, Adenovirus 2, Simian Virus 40 (SV40), and humancytomegalovirus. DNA sequences derived from the SV40 viral genome, forexample, SV40 origin, early and late promoter, enhancer, splice, andpolyadenylation sites may be used to provide the other genetic elementsrequired for expression of a heterologous DNA sequence. The early andlate promoters are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originor replication (Fiers et al., Nature 273:113, 1978). Smaller or largerSV40 fragments may also be used, provided the approximately 250 bpsequence extending from the Hind III site toward the BglI site locatedin the viral origin of replication is included.

Exemplary vectors can be constructed as disclosed by Okayama and Berg(Mol. Cell. Biol. 3:280, 1983). One useful system for stable high levelexpression of mammalian receptor cDNAs in C127 murine mammary epithelialcells can be constructed substantially as described by Cosman et al.(Mol. Immunol. 23:935, 1986). Vectors derived from retroviruses also maybe employed.

When secretion of the AcPL and/or IL-1Rrp1 protein from the host cell isdesired, the expression vector may comprise DNA encoding a signal orleader peptide. In place of the native signal sequence, a heterologoussignal sequence may be added, such as the signal sequence forinterleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195; the signalsequence for interleukin-2 receptor described in Cosman et al., Nature312:768 (1984); the interleukin-4 signal peptide described in EP367,566; the type I interleukin-1 receptor signal peptide described inU.S. Pat. No. 4,968,607; and the type II interleukin-1 receptor signalpeptide described in EP 460,846.

The present invention provides a process for preparing the recombinantproteins of the present invention, comprising culturing a host celltransformed with an expression vector comprising a DNA sequence thatencodes said protein under conditions that promote expression. Thedesired protein is then purified from culture media or cell extracts.The desired protein may be AcPL, IL-1Rrp1 or the heterodimeric receptor,for example. Cell-free translation systems could also be employed toproduce the desired protein using RNA derived from the novel DNA of thepresent invention.

As one example, supernatants from expression systems that secreterecombinant protein into the culture medium can be first concentratedusing a commercially available protein concentration filter, forexample, an Amicon or Millipore Pellicon ultrafiltration unit. Followingthe concentration step, the concentrate can be applied to a suitablepurification matrix. For example, a suitable affinity matrix cancomprise IL-18. An IL-18 affinity matrix may be prepared by couplingrecombinant human IL-18 to cyanogen bromide-activated Sepharose(Pharmacia) or Hydrazide Affigel (Biorad), according to manufacturer'srecommendations. Sequential immunopurification using antibodies bound toa suitable support is preferred. Proteins binding to an antibodyspecific for AcPL are recovered and contacted with antibody specific forIL-1Rrp1 on an insoluble support. Proteins immunoreactive with bothantibodies may thus be identified and isolated.

Alternatively, an anion exchange resin can be employed, for example, amatrix or substrate having pendant diethylaminoethyl (DEAE) groups. Thematrices can be acrylamide, agarose, dextran, cellulose or other typescommonly employed in protein purification. Alternatively, a cationexchange step can be employed. Suitable cation exchangers includevarious insoluble matrices comprising sulfopropyl or carboxymethylgroups. Sulfopropyl groups are preferred. One or more reversed-phasehigh performance liquid chromatography (RP-HPLC) steps employinghydrophobic RP-HPLC media, e.g., silica gel having pendant methyl orother aliphatic groups, can be employed to further purify a fusionprotein.

Some or all of the foregoing purification steps, in variouscombinations, can be employed to provide an essentially homogeneousrecombinant protein. Recombinant cell culture enables the production ofthe fusion protein free of those contaminating proteins which may benormally associated with IL-1Rrp1 or AcPL as they are found in nature intheir respective species of origin, e.g., on the surface of certain celltypes.

The foregoing purification procedures are among those that may beemployed to purify non-recombinant receptors of the present invention aswell. When linking procedures that may produce homodimers(IL-1Rrp1-linker-IL-1Rrp1 and AcPL-linker-AcPL) are employed,purification procedures that separate the heterodimer from suchhomodimers are employed. An example of such a procedure is sequentialimmunopurification as discussed above. In one embodiment, AcPL(recombinant or non-recombinant) is purified such that no bandscorresponding to other (contaminating) proteins are detectable bySDS-PAGE.

Recombinant protein produced in bacterial culture is usually isolated byinitial extraction from cell pellets, followed by one or moreconcentration, salting-out, aqueous ion exchange or size exclusionchromatography steps. Finally, high performance liquid chromatography(HPLC) can be employed for final purification steps. Microbial cellsemployed in expression of recombinant fusion proteins can disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents.

Fermentation of yeast which express fusion proteins as a secretedprotein greatly simplifies purification. Secreted recombinant proteinresulting from a large-scale fermentation can be purified by methodsanalogous to those disclosed by Urdal et al. (J. Chromatog. 296:171,1984), involving two sequential, reversed-phase HPLC steps forpurification of a recombinant protein on a preparative HPLC column.

The DNA or amino acid sequences of IL-1Rrp1 or AcPL may vary from thosepresented in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7. Dueto the known degeneracy of the genetic code, there can be considerablevariation in nucleotide sequences encoding the same amino acid sequence.In addition, DNA sequences capable of hybridizing to the native DNAsequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 undermoderately stringent or highly stringent conditions, and which encode abiologically active IL-1Rrp1 or AcPL polypeptide, are also considered tobe IL-1Rrp1-encoding or AcPL-encoding DNA sequences, in the context ofthe present invention. Such hybridizing sequences include but are notlimited to variant sequences such as those described below, and DNAderived from other mammalian species.

Moderately stringent conditions include conditions described in, forexample, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nded., Vol. 1, pp 1.101-104, Cold Spring Harbor Laboratory Press, 1989.Conditions of moderate stringency, as defined by Sambrook et al.,include use of a prewashing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH8.0) and hybridization conditions of about 55° C., 5×SSC, overnight.Highly stringent conditions include higher temperatures of hybridizationand washing. The skilled artisan will recognize that the temperature andwash solution salt concentration may be adjusted as necessary accordingto factors such as the length of the probe, wherein said conditionsinclude hybridization at 68° C. followed by washing in 0.1×SSC/0.1% SDSat 63-68° C. In another embodiment, the present invention provides aheterodimeric receptor comprising AcPL and IL-1Rrp1, wherein the AcPLand the IL-1Rrp1 are encoded by DNA that hybridizes to the DNA of SEQ IDNO:1 or SEQ ID NO:5, or SEQ ID NO:3 or SEQ ID NO:7, respectively, undermoderately or highly stringent conditions.

Further, certain mutations in a nucleotide sequence which encodes AcPLor IL-1Rrp1 will not be expressed in the final protein product. Forexample, nucleotide substitutions may be made to enhance expression,primarily to avoid secondary structure loops in the transcribed mRNA(see EP 75,444A). Other alterations of the nucleotide sequence may bemade to provide codons that are more readily translated by the selectedhost, e.g., the well-known E. coli preference codons for E. coliexpression.

The amino acid sequence of native IL-1Rrp1 or AcPL may be varied bysubstituting, deleting, adding, or inserting one or more amino acids toproduce a IL-1Rrp1 or AcPL variant. Variants that possess the desiredbiological activity of the native IL-1Rrp1 and AcPL proteins may beemployed in the receptor of the present invention. Assays by which thebiological activity of variant proteins may be analyzed are described inthe examples below. Biologically active IL-1Rrp1 polypeptides arecapable of binding IL-18. The desired biological activity of the AcPLpolypeptides disclosed herein is the ability to enhance the binding ofIL-18 when AcPL is joined to IL-1Rrp1, compared to the level of IL-18binding to IL-1Rrp1 alone.

Alterations to the native amino acid sequence may be accomplished by anyof a number of known techniques. For example, mutations can beintroduced at particular loci by synthesizing oligonucleotidescontaining a mutant sequence, flanked by restriction sites enablingligation to fragments of the native sequence. Following ligation, theresulting reconstructed sequence encodes an analog having the desiredamino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered gene having particularcodons altered according to the substitution, deletion, or insertionrequired. Exemplary methods of making the alterations set forth aboveare disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene37:73, 1985); Craig (BioTechniques, Jan. 1985, 12-19); Smith et al.(Genetic Engineering: Principles and Methods, Plenum Press, 1981); U.S.Pat. No. 4,518,584, and U.S. Pat. No. 4,737,462, which are incorporatedby reference herein.

Bioequivalent variants of AcPL and IL-1Rrp1 may be constructed by, forexample, making various substitutions of amino acid residues or deletingterminal or internal amino acids not needed for biological activity. Inone embodiment of the invention, the variant amino acid sequence is atleast 80% identical, preferably at least 90% identical, to the nativesequence. Percent similarity may be determined, for example, bycomparing sequence information using the GAP computer program, version6.0, available from the University of Wisconsin Genetics Computer Group(UWGCG). The GAP program utilizes the alignment method of Needleman andWunsch (J. Mol. Biol. 48:443, 1970), as revised by Smith and Waterman(Adv. Appl. Math. 2:482, 1981). Briefly, the GAP program definessimilarity as the number of aligned symbols (i.e., nucleotides or aminoacids) which are similar, divided by the total number of symbols in theshorter of the two sequences. The preferred default parameters for theGAP program include: (1) a unary comparison matrix (containing a valueof 1 for identities and 0 for non-identities) for nucleotides, and theweighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas ofProtein Sequence and Structure, National Biomedical Research Foundation,pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

Generally, substitutions should be made conservatively; i.e., the mostpreferred substitute amino acids are those having physiochemicalcharacteristics resembling those of the residue to be replaced. Examplesof conservative substitutions include substitution of one aliphaticresidue for another, such as Ile, Val, Leu, or Ala for one another, orsubstitutions of one polar residue for another, such as between Lys andArg; Glu and Asp; or Gln and Asn. Other such conservative substitutions,for example, substitutions of entire regions having similarhydrophobicity characteristics, are well known.

Cysteine residues can be deleted or replaced with other amino acids toprevent formation of unnecessary or incorrect intramolecular disulfidebridges upon renaturation. Hydrophilic amino acids may be substitutedfor hydrophobic amino acids in the transmembrane region and/orintracellular domain of IL-1Rrp1 and AcPL to enhance water solubility ofthe proteins.

Adjacent dibasic amino acid residues may be modified to enhanceexpression in yeast systems in which KEX2 protease activity is present.EP 212,914 discloses the use of site-specific mutagenesis to inactivateKEX2 protease processing sites in a protein. KEX2 protease processingsites are inactivated by deleting, adding or substituting residues toalter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence ofthese adjacent basic residues. These amino acid pairs, which constituteKEX2 proteases processing sites, are found at residues 98-99, 323-333,333-334, 472-473 and 475-476 of the AcPL protein of SEQ ID NO:2. TheseKEX2 sites are found at positions 113-114, 314-315, 364-365, 437-438,and 465-466 of the IL-1Rrp1 protein of SEQ ID NO:4. Lys-Lys pairings areconsiderably less susceptible to KEX2 cleavage, and conversion ofArg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferredapproach to inactivating KEX2 sites.

The present invention also includes proteins with or without associatednative-pattern glycosylation. Expression of DNAs encoding the fusionproteins in bacteria such as E. coli provides non-glycosylatedmolecules. Functional mutant analogs having inactivated N-glycosylationsites can be produced by oligonucleotide synthesis and ligation or bysite-specific mutagenesis techniques. These analog proteins can beproduced in a homogeneous, reduced-carbohydrate form in good yield usingyeast expression systems. N-glycosylation sites in eukaryotic proteinsare characterized by the amino acid triplet Asn-A₁-Z, where A1 is anyamino acid except Pro, and Z is Ser or Thr. In this sequence, asparagineprovides a side chain amino group for covalent attachment ofcarbohydrate.

The AcPL amino acid sequence in SEQ ID NO:2 contains 4 suchN-glycosylation sites, all found in the extracellular domain, at aminoacids 21-23, 119-121, 152-254 and 345-347. The extracellular domain ofIL-1Rrp1 comprises N-glycosylation sites at positions 91-93, 102-104,150-153, 168-170, 197-199, 203-205, 236-238, and 297-299 of SEQ ID NO:4.Such a site can be eliminated by substituting another amino acid for Asnor for residue Z, deleting Asn or Z, or inserting a non-Z amino acidbetween A₁ and Z, or an amino acid other than Asn between Asn and A₁.Known procedures for inactivating N-glycosylation sites in proteinsinclude those described in U.S. Pat. No. 5,071,972 and EP 276,846.

Variants of the receptor proteins of the present invention also includevarious structural forms of the primary protein which retain biologicalactivity. Due to the presence of ionizable amino and carboxyl groups,for example, a receptor protein may be in the form of acidic or basicsalts, or may be in neutral form. Individual amino acid residues mayalso be modified by oxidation or reduction.

The primary amino acid structure also may be modified by formingcovalent or aggregative conjugates with other chemical moieties, such asglycosyl groups, lipids, phosphate, acetyl groups and the like. Covalentderivatives are prepared by linking particular functional groups toamino acid side chains or at the N- or C-termini. Other derivatives ofthe receptor protein within the scope of this invention include covalentor aggregative conjugates of the receptor protein with other proteins orpolypeptides, such as by synthesis in recombinant culture as N- orC-terminal fusions. For example, the conjugated polypeptide may be asignal (or leader) polypeptide sequence at the N-terminal region of theprotein which co-translationally or post-translationally directstransfer of the protein from its site of synthesis to its site offunction inside or outside of the cell membrane or wall (e.g., the yeastα-factor leader).

Peptides may be fused to the desired protein (e.g., via recombinant DNAtechniques) to facilitate purification or identification. Examplesinclude poly-His or the Flag® peptide (Hopp et al., Bio/Technology6:1204, 1988, and U.S. Pat. No. 5,011,912). The Flag® peptide is highlyantigenic and provides an epitope reversibly bound by a specificmonoclonal antibody, enabling rapid assay and facile purification ofexpressed recombinant protein. Expression systems useful for fusing theFlag® octapeptide to the N- or C-terminus of a given protein areavailable from Eastman Kodak Co., Scientific Imaging Systems Division,New Haven, Conn., as are monoclonal antibodies that bind theoctapeptide.

Dimer IL-18 receptor complexes that include naturally occurring variantsof IL-1Rrp1 and AcPL are also encompassed by the present invention.Examples of such variants are proteins that result from alternative mRNAsplicing events or from proteolytic cleavage of the AcPL and IL-1Rrp1protein, wherein the desired biological activity is retained.Alternative splicing of mRNA may yield a truncated but biologicallyactive AcPL and IL-1Rrp1 protein, such as a naturally occurring solubleform of the protein. Variations attributable to proteolysis include, forexample, differences in the N-or C-termini upon expression in differenttypes of host cells, due to proteolytic removal of one or more terminalamino acids from the AcPL or IL-1Rrp1 protein (generally from 1-5terminal amino acids).

The present invention also provides a pharmaceutical compositioncomprising a receptor protein of the present invention with aphysiologically acceptable carrier or diluent. Such carriers anddiluents will be nontoxic to recipients at the dosages andconcentrations employed. Such compositions may, for example, comprisethe receptor protein in a buffered solution, to which may be addedantioxidants such as ascorbic acid, low molecular weight (less thanabout ten residues) polypeptides, proteins, amino acids, carbohydratesincluding glucose, sucrose or dextrins, chelating agents such as EDTA,glutathione and other stabilizers and excipients. The receptor of thepresent invention may be administered by any suitable method in a mannerappropriate to the indication, such as intravenous injection, localadministration, continuous infusion, sustained release from implants,etc.

Heterodimeric receptors of the present invention are useful as an IL-18binding reagent. This receptor, which preferably comprises soluble AcPLand soluble IL-1Rrp1, has applications both in vitro and in vivo. Thereceptors may be employed in in vitro assays, e.g., in studies of themechanism of transduction of the biological signal that is initiated bybinding of IL-18 to this receptor on a cell. Such receptors also couldbe used to inhibit a biological activity of IL-18 in various in vitroassays or in vivo procedures. In one embodiment of the invention, theinventive receptor is administered to bind IL-18, thus inhibitingbinding of the IL-18 to endogenous cell surface receptors. Biologicalactivity mediated by such binding of IL18 to the cells thus is alsoinhibited.

IL-1Rrp1 alone binds IL-18, but with relatively low affinity (Torigoe etal. J. Biol. Chem 272:2573, 1997). Receptors of the present invention,produced by cells co-transfected with AcPL and IL-1Rrp1-encoding DNA,for example, bind IL-18 with high affinity. Such receptors of thepresent invention may be employed when inhibition of an IL-18-mediatedactivity is desired. In addition, use of the receptors of the presentinvention in vitro assays offers the advantage of allowing one todetermine that the assay results are attributable to binding of IL18.

In one embodiment of the invention, a heterodimeric receptor comprisingAcPL and IL-1Rrp1 is administered in vivo to inhibit a biologicalactivity of IL-18. IL-18 is known to mediate NFκB activity and acts as astimulator of Th1 cell growth and differentiation, and is a potentinducer of γ-interferon production from Th1 cells. IL-18 also enhancesNK cell killing activity and has been implicated in septic shock, liverdestruction, and diabetes. When these or other biological effects ofIL-18 are undesirable, a receptor of the present invention may beadministered to bind IL-18 and ameliorate the effects of IL-18 activity.

The inventive receptor may be administered to a patient in atherapeutically effective amount to treat a disorder mediated by IL-18.A disorder is said to be mediated by IL-18 when IL-18 causes (directlyor indirectly) or exacerbates the disorder. Soluble receptor proteinscan be used to competitively bind to IL-18, thereby inhibiting bindingof IL-18 to endogenous cell surface receptors.

Heterodimeric receptors comprising AcPL linked to IL-1Rrp1 also find usein assays for biological activity of IL-18 proteins, which biologicalactivity is measured in terms of binding affinity for the receptor. Toillustrate, the receptor may be employed in a binding assay to measurethe biological activity of an IL-18 fragment, variant, or mutein. Thereceptor is useful for determining whether biological activity of IL-18is retained after modification of an IL-18 protein (e.g., chemicalmodification, mutation, etc.). The binding affinity of the modifiedIL-18 protein for the receptor is compared to that of an unmodified IL18protein to detect any adverse impact of the modification on biologicalactivity. Biological activity thus can be assessed before the modifiedprotein is used in a research study or assay, for example.

The heterodimeric receptors also find use as reagents that may beemployed by those conducting “quality assurance” studies, e.g., tomonitor shelf life and stability of IL-18 proteins under differentconditions. The receptors may be used to confirm biological activity (interms of binding affinity for the receptor) in IL-18 proteins that havebeen stored at different temperatures, for different periods of time, orwhich have been produced in different types of recombinant expressionsystems, for example.

The following examples are provided to illustrate certain embodiments ofthe invention, and are not to be construed as limiting the scope of theinvention.

EXAMPLES Example 1 In Vitro Precipitation Experiments

In order to determine whether AcPL, IL-1Rrp1 or combinations of the twopolypeptides bind IL-18, several Fc fusion proteins were prepared andtested as follows. An expression vector encoding a soluble AcPL/Fcfusion protein, which comprised a truncated extracellular domain of AcPLfused to the N-terminus of an Fc region polypeptide derived from anantibody, was constructed as follows. A recombinant expression vectorcomprising AcPL DNA in vector pDC304, was PCR amplified utilizingprimers containing the desired in-frame restriction sites on the 5′ and3′ ends. The resulting fragment, which includes the 5′ end of the AcPLDNA, terminating at nucleotide 1551 of SEQ ID NO:1, with introduced SalIand BglII sites at the 5′ and 3′ ends, respectively, was isolated, byconventional techniques.

A recombinant vector designated pDC412-hIgG1Fc comprises the Fcpolypeptide-encoding cDNA (—H—CH₂—CH₃ only). Vector pDC412-hIgG1Fc wasdigested with the restriction enzymes SalI and BglII, which cleave inthe polylinker region of the vector, upstream of the Fcpolypeptide-encoding cDNA.

The AcPL-encoding DNA fragment isolated above was ligated into aSalI/BglII-digested pDC412-hIgG1Fc such that the Fc polypeptide DNA wasfused to the 3′ end of the AcPL DNA. The resulting expression vectorencoded a fusion protein comprising amino acids 1-356 of the AcPLsequence of SEQ ID NO:2, followed by the H—CH₂—CH₃ region of hIgG1Fc.

An expression vector encoding a soluble human IL-1Rrp1/Fc fusion proteinwas constructed as follows. A recombinant vector that includes IL-1Rrp1cDNA was PCR amplified with gene-specific primers containing the desiredrestriction sites. The resulting fragment, which includes the 5′ end ofIL-1Rrp1 was isolated by conventional techniques. This IL-1Rrp1fragment, digested with Asp718 and BglII, was combined with the hIgG1Fcfragment described above and digested with BglII and NotI. The resultingdigest fragments were ligated to pDC304 digested with Asp718 and Not I.The resulting IL-1Rrp1/Fc fusion protein encoded by the recombinantvector comprises (from N- to C-terminus) amino acids 1-329 of SEQ IDNO:4, followed by the H—CH₂—CH₃ region of hIgG1/Fc.

In one sample set, COS-7 cells were transfected with a pDC206 control orpDC206-IL-18 vector. In another sample set of transfected COS-7 cells,the Fc fusion vectors described above were transfected. The total sampleset was as follows: Sample Cells transfected with vector(s) encoding: Aempty pDC206 expression vector (control) B pDC206hIL-18 1 pDC409(control) 2 IL-1Rrp1/Fc 3 AcPL/Fc 4 AcPL/Fc and IL-1Rrp1/FcTwo days post transfection, samples A and B were starved 1 hour incys/met-free medium, then labeled with [³⁵S-cys][³⁵S-met]-containingmedium for 6 hours. Supernatants were removed, subjected tocentrifugation, and adjusted to 0.4M NaCl/1.0% Triton X-100 in thepresence of protease inhibitors. The supernatants from cells transfectedwith the Fc fusion proteins of Samples 1-4 were removed at 2 days posttransfection and centrifuged. Each Fc fusion supernatant was combinedwith either a) vector-transfected: or, b) IL-18 transfected ³⁵S-labeledsupernatants. Purified IL-1Rrp1/Fc-receptor protein was added to anotherportion of the radiolabeled supernatant as a control. ProteinG-Sepharose was added to each experimental sample and precipitationswere carried out overnight at 4° C. Then the samples were washedextensively in a 0.4M NaCl, 0.05% SDS, 1.0% NP-40 buffer and separatedby electrophoresis in a 4-20% Tris-Glycine gel. The gel was fixed,amplified, dried and exposed to film. In order to assess total levels ofprotein and take into account unlabeled Fc-fusion proteins, a portion ofeach precipitate was analyzed on a separate 4-20% Tris-Glycine gel andsilver stained.

Supernatants from cell samples 1-4 did not noticeably precipitate anyproteins in the 10-30 Kd range from the control supernatant (Sample A).IL-18 (Sample B) was precipitated by supernatant from cell sample 2(IL-1Rrp1Fc) but not by supernatant from cell sample 1 (control) or cellsample 3 (AcPL/Fc). Significantly more IL-18 was precipitated bysupernatant from all sample 4 that was obtained from the cotransfectionof IL-1Rrp1/Fc and AcPL/Fc.

Thus, IL-1Rrp1 is able to bind IL-18; AcPL is not able to bind IL-18;and, coexpressed IL-1Rrp1 and AcPL are able to bind Il-18 and thecoexpressed proteins exhibit higher levels of binding of IL-18 thanIL-1Rrp1alone. The silver stained gel shows that there is no moreIL-1Rrp1 in supernatants transfected with Il-1Rrp1 and AcPL as comparedto supernatants transfected with IL-1Rrp1 alone. This rules out thepossibility that there is more IL-1Rrp1 expressed in these samples. Theresults indicate that the IL-18 binding affinity of an IL-1Rrp1 /AcPLdimer is greater than the affinity of IL-1Rrp1 alone.

Example 2 Induction of NFκB Activity

In order to determine the roles of IL-1Rrp1 and AcPL in IL-18 signaling,AcPL, IL-1Rrp1, and a combination of IL-1Rrp1 and AcPL wereoverexpressed in COS cells and S49.1 cells and the effect of IL-18stimulation on NFκB activation was assessed.

COS-7 cells were transfected by the DEAE/Dextran method in a 12-wellformat. Each well was transfected with a total of 200 ng of theappropriate expression vector(s) and 800 ng of a NFκB-Luc reporterplasmid, which contains 3 NFκB sites mediating luciferase expression.Approximately 10⁷ S49.1 cells were transfected by electroporation in 0.7mL with 40 μg of the NFκB-Luc reporter plasmid, and a total of 20 μg ofthe appropriate expression vector(s). Electroporations were performed at960 μF and 320V.

The cells were incubated for 2 days, and then stimulated with 40 ng/mLof IL-18 (purchased from PeproTech) for 4 hours. Cells were washed,lysed, and assayed for luciferase activity using Luciferase AssayReagents (purchased from Promega Corp.) according to the manufacturer'sinstructions.

COS7 or S49.1 cells that were transfected with control vector alone,vector encoding mIL-1Rrp1 alone, or vector encoding mAcPL alone were notresponsive to mIL-18 stimulation. Furthermore, S49.1 cells transfectedwith mAcPL were not responsive to mIL-18 signaling when the transfectionwas in combination with an expression vector encoding mIL-1R type I ormIL-1RAcP. However, cells cotransfected with mAcPL and mIL-1Rrp1 andstimulated with mIL-18 showed an increase in NFκB DNA binding activitythat was 10 fold in COS cells and 300 fold in S49.1 cells. COS7 cellstransfected with hIL-1Rrp1 displayed no response to hIL-18 stimulation,while COS7 cells transfected with hAcPL alone and stimulated with hIL-18showed an 8 fold increase in NFκB activity. This is attributed to theassociation of hAcPL with monkey IL-1Rrp1 endogenous to COS7 cells.Overexpression of hIL-1Rrp1 with hAcPL did not augment the stimulationof NFκB activity in response to hIL-18 over that seen in cellsoverexpressing hAcPL alone. This dramatic enhancement of NFκB activityindicates that AcPL and IL-1Rrp1 are subunits of the IL-18 receptor andcooperate to induce NFκB signaling in response to EL-18 stimulation.

Example 3 Preparing AcPL and IL-1Rrp1 Antibody Heavy and Light ChainFusion Proteins

The following describes preparing fusion proteins that include AcPL andIL-1Rrp1 fused to an antibody heavy chain and antibody light chainpolypeptide.

First, an expression vector encoding the entire constant region of humanIgG1 with a linker region upstream is constructed. Such an expressionvector facilitates the creation of fusion protein-encoding plasmids. PCRtechniques are utilized to amplify the above mentioned IgG1 constantregion with primers containing an upstream BglII site and a downstreamNotI site. The resulting PCR generated fragment is digested, purified,and ligated to pDC412 which is digested with BglII and NotI. ThepDC412-hIgG1 expression vector is then digested with SalI and BglII.

Next an expression vector containing the Ig κ constant domain precededby a linker region and followed by a linker region and poly-His domainis prepared. The poly-His domain advantageously aids in the proteinpurification process. PCR techniques are utilized to amplify theconstant region with primers containing a BglII-NotI fragment. Theresulting PCR generated fragment is digested, purified, and ligated topDC412. Soluble receptors of interest can be cloned upstream byutilizing the unique SalI and BglII sites.

To prepare an IL-1Rrp1-Cκ expression vector, the extracellular domain ofIL-1Rrp1 is PCR amplified using primers containing SalI (5′) and BglII(3′) restriction sites. This purified and digested PCR product isligated to SalI/BglII digested the pDC412-Ig κ expression vector tocreate an in-frame construct that encodes a fusion protein linking thesoluble portion of IL-1Rrp1 to the constant region of Cκ.

To prepare an IL-1Rrp1-hIgG1 expression vector the SalI/BglIIrestriction fragment encoding soluble IL-1Rrp1 is removed from IL-1Rrp1-Cκ and ligated to pDC412-hIgG1 which has been digested with the samerestriction enzymes. Since both vectors contain the BglII site in thesame reading frame, this will readily generate a fusion between solubleIL-1Rrp1 and hIgG1.

To prepare an AcPL-Cκ expression vector, the extracellular domain ofAcPL is PCR amplified using primers containing SalI (5′) and BglII (3′)restriction sites. This purified and digested PCR product then isligated to SalI/BglII digested pDC412-Cκ to create an in-frame fusionprotein linking the soluble portion of AcPL to the constant region ofCκ.

To prepare an AcPL-hIgG1 expression vector the SalI/BglII restrictionfragment encoding soluble AcPL is removed from AcPL-cκ and ligated topDC412-hIgG1 which has been digested with the same restriction enzymes.Since both vectors contain the BglII site in the same reading frame,this will readily generate a fusion between soluble AcPL and hIgG1.

COS-7 cells are transfected with the above described fusion vectors. Thecells are cultured and the fusion proteins are collected as described inExample 1.

Example 4 Inhibition of an IL18 Induced NFκB Activity

Cos7 cells were transiently transfected in 12-well plates with 10 ngeach of mIL-1Rrp1 and mAcPL expression vectors, and 50 ng of a3×NFκB-Luciferase reporter plasmid per well. Two days post-transfection,cells were stimulated with 10 ng/ml mIL-18 (purchased from Peprotech) inthe presence of increasing amounts of various receptor-Fc fusionproteins. mIL-18 was preincubated with the proteins for 20 min at roomtemperature prior to addition to cells. The amount of Fc protein wastitrated from 1 ug/ml to 50 ug/ml. Cells were stimulated 4 hours, thenlysed and luciferase activity was assessed using the Promega LuciferaseAssay Reagents.

Preincubation of IL-18 with mIL-1Rrp1-Fc, mIL-1Rrp1 -FlagpolyHis, ormAcPL-Fc had no significant effect on induction of NFκB at any of theconcentrations tested. In contrast, incubation of IL-18 with aheterogeneous mIL-1Rrp1-Fc+mAcPL-Fc protein mixture (consisting ofhomodimers of mIL-1Rrp1-Fc, homodimers of mAcPL-Fc, and heterodimericmIL-1Rrp1-Fc/mAcPL-Fc molecules) resulted in a dose-dependent inhibitionof NFκB induction. Uninduced cells displayed 3×10e3 RLU, and cellsstimulated with mIL-18 in the absence of any receptor-Fc proteindisplayed 25×10e3 RLU. Maximal inhibition of NFκB induction was observedwith 20 ug/ml and 50 ug/ml of mIL-1R-rp1-Fc+mAcPL-Fc protein mixture,which resulted in 6×10e3 RLU, representing an 87% inhibition of IL-18activity.

1-7. (canceled)
 8. An isolated protein comprising at least one firstpolypeptide linked to at least one second polypeptide, wherein saidfirst polypeptide binds to IL-18, and further wherein said firstpolypeptide is encoded by a DNA selected from the group consisting of:a) DNA comprising the coding region of the nucleotide sequence presentedin SEQ ID NO:7; b) DNA that is complementary to DNA capable ofhybridizing to the DNA of (a) under highly stringent conditions(hybridization at 68° C. followed by washing in 0.1×SSC/0.1% SDS at63-68° C.); and, c) DNA that encodes a polypeptide comprising the aminoacid sequence presented in SEQ ID NO:8; and, wherein said secondpolypeptide is encoded by a DNA selected from the group consisting of:a′) DNA comprising the coding region of the nucleotide sequencepresented in SEQ ID NO:5; b′) DNA that is complementary to DNA capableof hybridizing to the DNA of (a′) under highly stringent conditions(hybridization at 68° C. followed by washing in 0.1×SSC/0.1% SDS at63-68° C.); and c′) DNA that encodes a polypeptide comprising the aminoacid sequence presented in SEQ ID NO:6, wherein the isolated proteinbinds IL-18 with an affinity greater than that of the first polypeptidealone.
 9. An isolated protein comprising at least one first polypeptidelinked to at least one second polypeptide, wherein said firstpolypeptide binds to IL-18, and further wherein said first polypeptideis encoded by a DNA selected from the group consisting of: a) DNAencoding a polypeptide comprising amino acids y-325 of SEQ ID NO:8,wherein y represents an integer between and including 1 and 19; and b)DNA that is complementary to DNA capable of hybridizing to the DNA of a)under highly stringent conditions (hybridization at 68° C. followed bywashing in 0.1×SSC/0.1% SDS at 63-68° C.); and wherein said secondpolypeptide is encoded by a DNA selected from the group comprising: a′)DNA encoding a polypeptide comprising amino acids x-356 of SEQ ID NO:6,wherein x is an integer between and including 1 and 15; and b′) DNA thatis complementary to DNA capable of hybridizing to the DNA of a′) underhighly stringent conditions (hybridization at 68° C. followed by washingin 0.1×SSC/0.1% SDS at 63-68° C.), wherein the isolated protein bindsIL-18 with an affinity greater than that of the first polypeptide alone.10. An isolated protein comprising at least one first polypeptide linkedto at least one second polypeptide, wherein the first polypeptidecomprises amino acids 19 through 325 of SEQ ID NO:8 and the second polypeptide comprises amino acids 15 through 356 of SEQ ID NO:6.
 11. Theisolated protein of claim 10, wherein the first polypeptide and secondpolypeptide are linked via a peptide linker.
 12. An isolated proteincomprising at least one first polypeptide linked to at least one secondpolypeptide, wherein the second polypeptide comprises a polypeptide thatis at least 80% identical to a polypeptide having amino acids x-356 ofSEQ ID NO:6, wherein x is an integer between and including 1 and 15; andwherein the first polypeptide binds to IL-18, and further wherein saidfirst polypeptide comprises a polypeptide that is at least 80% identicalto a polypeptide having amino acids y-325 of SEQ ID NO:8, wherein yrepresents an integer between and including 1 and 19; and, furtherwherein the isolated protein binds IL-18 with an affinity greater thanthat of the first polypeptide alone.
 13. The isolated protein of claim12, wherein the first polypeptide comprises a polypeptide having aminoacids y-325 of SEQ ID NO:8, and the second polypeptide comprises apolypeptide having amino acids x-356 of SEQ ID NO:6.
 14. A proteinhaving a formula selected from the group consisting of:R₁-L₁:R₂₋-L₂, R₂-L₂:R₁-L₁, R₁-L₂:R₂-L₁, R₂-L₁:R₁-L₂,R₁-L₁:R₂₋-L₂/R₂-L₂:R₁-L₁, and, R₁-L₂:R₂-L₁/R₂-L₁:R₁-L₂ wherein L₁ is animmunoglobulin heavy chain fragment; L₂ is an immunoglobulin light chainfragment; : is a linkage between a heavy chain and light chain antibodyregion, / is a linkage between a light chain and a heavy chain antibodyregion; and wherein R₂ is selected from the group consisting of: a) apolypeptide comprising amino acids y-325 of SEQ ID NO:8, wherein yrepresents an integer between and including 1 and 19; b) a polypeptidethat is at least 90% identical to the polypeptide of a); and, c) afragment of the polypeptide of a), and further wherein R₂ binds IL-18;wherein said R₁ is selected from the group consisting of: a′) apolypeptide comprising amino acids x-356 of SEQ ID NO:6, wherein xrepresents an integer between and including 1 and 15; b′) a polypeptidethat is at least 90% identical to the polypeptide of a′); and c′) afragment of the polypeptide of a′), and further wherein the proteinbinds IL-18 with an affinity greater than that of R₂ alone.
 15. Aheterodimeric protein comprising a first fusion polypeptide and a secondfusion polypeptide, the first fusion polypeptide comprising an antibodylight chain polypeptide attached to the C-terminus of a soluble firstpolypeptide or a soluble second polypeptide, and the second fusionpolypeptide comprising an antibody heavy chain polypeptide attached tothe C-terminus of a soluble first polypeptide or of a soluble secondpolypeptide, wherein said first fusion polypeptide is linked to saidsecond fusion polypeptide via disulfide bonds between the heavy chainand light chain polypeptides; and, wherein the soluble first polypeptideis selected from the group consisting of: a) polypeptides comprisingamino acids y-325 of SEQ ID NO:8, wherein y represents an integerbetween and including 1 and 19; and, b) polypeptides comprising afragment of a), further wherein the first soluble polypeptide bindsIL-18; and wherein the soluble second polypeptide is selected from thegroup consisting of: a′) polypeptides comprising amino acids x-356 ofSEQ ID NO:6, wherein x represents an integer between and including 1 and15; and b′) polypeptides comprising a fragment of a′), wherein thefragment of a′), in combination with soluble first polypeptide bindsIL-18 with an affinity greater than that of the soluble firstpolypeptide alone.
 16. A protein having a formula selected from thegroup consisting of:R₁-L₁:R₂₋-L₂, R₂-L₂:R₁-L₁, R₁-L₂:R₂-L₁, R₂-L₁:R₁-L₂,R₁-L₁: R₂₋-L₂/R₂-L₂: R₁-L₁, and, R₁-L₂: R₂-L₁/R₂-L₁:R₁-L₂ wherein L₁comprises an immunoglobulin heavy chain fragment; L₂ comprises animmunoglobulin light chain fragment; R₁ comprises a fragment of SEQ IDNO:6; R₂ comprises a fragment of SEQ ID NO:8; : is a linkage between aheavy chain and light chain antibody region, and / is a linkage betweena light chain and heavy chain antibody region, wherein the fragment ofSEQ ID NO:8 binds IL-18, and further wherein the protein binds IL-18with higher affinity than the fragment of SEQ ID NO:8 alone.
 17. Aprotein comprising at least one first polypeptide linked to at least onesecond polypeptide wherein the first polypeptide comprises a fragment ofamino acids 1-325 of SEQ ID NO:8 that binds IL-18 and the secondpolypeptide comprises a fragment of amino acids 1-356 of SEQ ID NO:6,wherein the protein binds IL-18 with a greater affinity than amino acids1-325 of SEQ ID NO:8 alone.
 18. A host cell genetically engineered toexpress a protein selected from the group consisting of the protein ofclaim 8, the protein of claim 9, the protein of claim 12, and theprotein of claim
 14. 19. A process of comprising culturing the host cellof claim 18 under conditions that promote expression of the protein. 20.A composition comprising a protein selected from the group consisting ofthe protein of claim 8, the protein of claim 9, the protein of claim 12,and the protein of claim
 14. 21. A method of inhibiting the effects ofIL-18 comprising administering an effective amount of the composition ofclaim 20 to a mammal.